The abnormal expressions of minichromosome maintenance protein 2 (MCM2) are closely related to the development of various kinds of cancers. We aimed to explore the functions and potential molecular mechanisms of MCM2 gene in cholangiocarcinoma (CCA) cell lines (Huh28 and RBE). First, the cell counting kit-8 (CCK-8), plate clone formation, transwell and invasion assays showed that MCM2 promotes the proliferation, migration and invasion of CCA cells. Flow cytometry assays showed that MCM2 significantly promotes the cell cycle, and inhibits the apoptosis of CCA cells. Further, by analyzing the RNA sequencing data of cholangiocarcinoma, we found that MCM2 gene is significantly negatively correlated with p53 signaling pathway. Quantitative real time polymerase chain reaction (qRT-PCR) and Western blotting (WB) assays confirmed that MCM2 in CCA cells significantly down-regulated the mRNA and protein expression levels of p53 and BAX, and up-regulated the mRNA and protein expression levels of BCL2 and CCND1. Flow cytometry, qRT-PCR and WB assays confirmed that MCM2 promotes CCA through p53 pathway. Finally, we found that MCM2 is up-regulated in CCA tissues compared to the matched non-tumor cholangiocarcinoma tissues, and the high expressions of MCM2 are significantly associated with the poor clinical outcomes of CCA patients. In conclusion, this study revealed that MCM2 promotes the development of CCA by reducing the p53 pathway, and its high expression levels predict poor prognosis in CCA patients. These results provide a theoretical basis for the development of new clinical diagnosis and treatment of cholangiocarcinoma in the future.
微小染色体维持蛋白2 (minichromosome maintenance protein 2, MCM2)的异常表达与多种恶性肿瘤的发生发展密切相关。为探索MCM2基因对胆管癌细胞肿瘤生物学行为的影响及其潜在的作用机制,本研究首先采用cell counting kit-8 (CCK-8)、平板细胞克隆形成、Transwell和Invasion实验证实,MCM2可促进胆管癌细胞的增殖、迁移和侵袭能力;流式细胞术检测结果显示,MCM2可加快细胞周期进程,抑制细胞凋亡。进一步通过对胆管癌组织RNA测序数据集的分析发现,MCM2基因与p53信号通路的激活显著负相关。采用实时定量PCR (quantitative real time polymerase chain reaction, qRT-PCR)和蛋白质免疫印迹(Western blotting, WB)实验发现,在胆管癌细胞中MCM2可显著下调p53和BAX的mRNA和蛋白表达水平,显著上调BCL2和CCND1的mRNA和蛋白表达水平。采用流式细胞术、qRT-PCR和WB实验进一步证实MCM2基因依赖于p53通路发挥影响胆管癌细胞周期和凋亡的功能。最后,通过分析MCM2的表达水平与胆管癌患者临床预后的相关性发现,MCM2在胆管癌组织中的表达显著高于癌旁组织,且其高表达与患者的不良预后显著相关。综上所述,本研究初步揭示MCM2可能通过抑制p53信号通路促进胆管癌的发生发展,并提示MCM2的高表达与胆管癌的不良预后显著相关,为未来胆管癌新的临床诊治措施的研发提供了理论依据。.
Keywords: MCM2; apoptosis; cell cycle; cholangiocarcinoma; invasion; migration; p53; proliferation.